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Molecular markers in plant

genome analysis
With the advent of molecular markers, a new generation of markers has been
introduced over the last two decades, which has revolutionized the entire scenario of
biological sciences. DNA based molecular markers have acted as versatile tools and have
found their own position in various fields like taxonomy, physiology, embryology, genetic
engineering, etc. They are no longer looked upon as simple DNA fingerprinting markers
in variability studies or as mere forensic tools. Ever since their development, they are
constantly being modified to enhance their utility and to bring about automation in the
process of genome analysis. The discovery of PCR (polymerase chain reaction) was a
landmark in this effort and proved to be an unique process that brought about a new
class of DNA profiling markers. This facilitated the development of marker-based gene
tags, map-based cloning of agronomically important genes, variability studies,
phylogenetic analysis, synteny mapping, marker-assisted selection of desirable
genotypes, etc. Thus giving new dimensions to concerted efforts of breeding and marker-
aided selection that can reduce the time span of developing new and better varieties and
will make the dream of super varieties come true. These DNA markers offer several
advantages over traditional phenotypic markers, as they provide data that can be
analysed objectively. In this article, DNA markers developed during the last two decades
of molecular biology research and utilized for various applications in the area of plant
genome analysis are reviewed.

PLANTS have always been looked upon as a key source of energy for
survival and evolution of the animal kingdom, thus forming a base for
every ecological pyramid. Over the last few decades plant genomics has
been studied extensively bringing about a revolution in this area.
Molecular markers, useful for plant genome analysis, have now become
an important tool in this revolution. In this article we attempt to review
most of the available DNA markers that can be routinely employed in
various aspects of plant genome analysis such as taxonomy, phylogeny,
ecology, genetics and plant breeding.

During the early period of research, classical strategies including

comparative anatomy, physiology and embryology were employed in
genetic analysis to determine inter- and intra-species variability. In the
past decade, however, molecular markers have very rapidly
complemented the classical strategies. Molecular markers include
biochemical constituents (e.g. secondary metabolites in plants) and
macromolecules, viz. proteins and deoxyribonucleic acids (DNA). Analysis
of secondary metabolites is, however, restricted to those plants that
produce a suitable range of metabolites which can be easily analysed and
which can distinguish between varieties. These metabolites which are
being used as markers should be ideally neutral to environmental effects
or management practices. Hence, amongst the molecular markers used,
DNA markers are more suitable and ubiquitous to most of the living
DNA-based molecular markers
Genetic polymorphism is classically defined as the simultaneous
occurrence of a trait in the same population of two or more discontinuous
variants or genotypes. Although DNA sequencing is a straightforward
approach for identifying variations at a locus, it is expensive and
laborious. A wide variety of techniques have, therefore, been developed in
the past few years for visualizing DNA sequence polymorphism. The term
DNA-fingerprinting was introduced for the first time by Alec Jeffrey in 1985
to describe bar-code-like DNA fragment patterns generated by multilocus
probes after electrophoretic separation of genomic DNA fragments. The
emerging patterns make up an unique feature of the analysed individual
and are currently considered to be the ultimate tool for biological
individualization. Recently, the term DNA fingerprinting/profiling is used to
describe the combined use of several single locus detection systems and
are being used as versatile tools for investigating various aspects of plant
genomes. These include characterization of genetic variability, genome
fingerprinting, genome mapping, gene localization, analysis of genome
evolution, population genetics, taxonomy, plant breeding, and diagnostics.

Properties desirable for ideal DNA markers

• Highly polymorphic nature

• Co-dominant inheritance (determination of homozygous and
heterozygous states of diploid organisms)
• Frequent occurrence in genome
• Selective neutral behaviour (the DNA sequences of any organism
are neutral to environmental conditions or management practices)
• Easy access (availability)
• Easy and fast assay
• High reproducibility
• Easy exchange of data between laboratories.

It is extremely difficult to find a molecular marker which would meet all

the above criteria. Depending on the type of study to be undertaken, a
marker system can be identified that would fulfil atleast a few of the
above characteristics.

Various types of molecular markers are utilized to evaluate DNA

polymorphism and are generally classified as hybridization-based markers
and polymerase chain reaction (PCR)-based markers. In the former, DNA
profiles are visualized by hybridizing the restriction enzyme-digested DNA,
to a labelled probe, which is a DNA fragment of known origin or sequence.
PCR-based markers involve in vitro amplification of particular DNA
sequences or loci, with the help of specifically or arbitrarily chosen
oligonucleotide sequences (primers) and a thermostable DNA polymerase
enzyme. The amplified fragments are separated electrophoretically and
banding patterns are detected by different methods such as staining and
autoradiography. PCR is a versatile technique invented during the mid-
1980s (ref. 3). Ever since thermostable DNA polymerase was introduced in
1988 (ref. 4), the use of PCR in research and clinical laboratories has
increased tremendously. The primer sequences are chosen to allow base-
specific binding to the template in reverse orientation. PCR is extremely
sensitive and operates at a very high speed. Its application for diverse
purposes has opened up a multitude of new possibilities in the field of
molecular biology. For simplicity, we have divided the review in two parts.
The first part is a general description of most of the available DNA marker
types, while the second includes their application in plant genomics and
breeding programmes.
Types and description of DNA markers
Single or low copy probes

Restriction fragment length polymorphism (RFLP). RFLPs are simply

inherited naturally occurring Mendelian characters. They have their origin
in the DNA rearrangements that occur due to evolutionary processes,
point mutations within the restriction enzyme recognition site sequences,
insertions or deletions within the fragments, and unequal crossing over.

In RFLP analysis, restriction enzyme-digested genomic DNA is

resolved by gel electrophoresis and then blotted6 on to a nitrocellulose
membrane. Specific banding patterns are then visualized by hybridization
with labelled probe. These probes are mostly species-specific single locus
probes of about 0.5–3.0 kb in size, obtained from a cDNA library or a
genomic library. The genomic libraries are easy to construct and almost
all sequence types are included; however, a large number of interspersed
repeats are found in inserts that detect a large number of restriction
fragments forming complex patterns. In plants, this problem is overcome
to some extent by using methylation-sensitive restriction enzyme PstI.
This helps to obtain low copy DNA sequences of small fragment sizes,
which are preferred in RFLP analysis. On the other hand cDNA libraries are
difficult to construct, however, they are more popular as actual genes are
analysed and they contain fewer repeat sequences. The selection of
appropriate source for RFLP probe varies, with the requirement of
particular application under consideration. Though genomic library probes
may exhibit greater variability than gene probes from cDNA libraries, a
few studies reveal the converse. This observation may be because cDNA
probes not only detect variation in coding regions of the corresponding
genes but also regions flanking genes and introns of the gene.

RFLP markers were used for the first time in the construction of
genetic maps by Botstein et al. RFLPs, being codominant markers, can
detect coupling phase of DNA molecules, as DNA fragments from all
homologous chromosomes are detected. They are very reliable markers in
linkage analysis and breeding and can easily determine if a linked trait is
present in a homozygous or heterozygous state in an individual, an
information highly desirable for recessive traits12. However, their utility
has been hampered due to the large amount of DNA required for
restriction digestion and Southern blotting. The requirement of radioactive
isotope makes the analysis relatively expensive and hazardous. The assay
is time-consuming and labour intensive and only one out of several
markers may be polymorphic, which is highly inconvenient especially for
crosses between closely-related species. Their inability to detect single
base changes restricts their use in detecting point mutations occurring
within the regions at which they are detecting polymorphism.

Restriction landmark genomic scanning (RLGS). This method, introduced

for the first time by Hatada et al.13, for genomic DNA analysis of higher
organisms, is based on the principle that restriction enzyme sites can be
used as landmarks. It employs direct labelling of genomic DNA at the
restriction site and two-dimensional (2D) electrophoresis to resolve and
identify these landmarks. The technique has proven its utility in genome
analysis of closely-related cultivars and for obtaining polymorphic markers
that can be cloned by spot target method. It has been used as a new
fingerprinting technique for rice cultivars.
RFLP markers converted in to PCR based-markers
Sequence-tagged sites (STS)
RFLP probes specifically linked to a desired trait can be converted
into PCR-based STS markers based on nucleotide sequence of the probe
giving polymorphic band pattern, to obtain specific amplicon. Using this
technique, tedious hybridization procedures involved in RFLP analysis can
be overcome. This approach is extremely useful for studying the
relationship between various species. When these markers are linked to
some specific traits, for example powdery mildew resistance gene or stem
rust resistance gene in barley, they can be easily integrated into plant
breeding programmes for marker-assisted selection of the trait of interest.

Allele-specific associated primers (ASAPs)

To obtain an allele-specific marker, specific allele (either in
homozygous or heterozygous state) is sequenced and specific primers are
designed for amplification of DNA template to generate a single fragment
at stringent annealing temperatures. These markers tag specific alleles in
the genome and are more or less similar to SCARs. Expressed sequence
tag markers (EST). This term was introduced by Adams et al. Such
markers are obtained by partial sequencing of random cDNA clones. Once
generated, they are useful in cloning specific genes of interest and
synteny mapping of functional genes in various related organisms. ESTs
are popularly used in full genome sequencing and mapping programmes
underway for a number of organisms and for identifying active genes thus
helping in identification of diagnostic markers. Moreover, an EST that
appears to be unique helps to isolate new genes. EST markers are
identified to a large extent for rice, Arabidopsis, etc. wherein thousands of
functional cDNA clones are being converted in to EST markers.

Single strand conformation polymorphism (SSCP)

This is a powerful and rapid technique for gene analysis particularly
for detection of point mutations and typing of DNA polymorphism. SSCP
can identify heterozygosity of DNA fragments of the same molecular
weight and can even detect changes of a few nucleotide bases as the
mobility of the single-stranded DNA changes with change in its GC content
due to its conformational change. To overcome problems of reannealing
and complex banding patterns, an improved technique called asymmetric-
PCR SSCP was developed, wherein the denaturation step was eliminated
and a large-sized sample could be loaded for gel electrophoresis, making
it a potential tool for high throughput DNA polymorphism. It was found
useful in the detection of heritable human diseases. In plants, however, it
is not well developed although its application in discriminating progenies
can be exploited, once suitable primers are designed for agronomically
important traits.

Multi locus probes

Repetitive DNA
A major step forward in genetic identification is the discovery that
about 30–90% of the genome of virtually all the species is constituted by
regions of repetitive DNA, which are highly polymorphic in nature. These
regions contain genetic loci comprising several hundred alleles, differing
from each other with respect to length, sequence or both and they are
interspersed in tandem arrays ubiquitously. The repetitive DNA regions
play an important role in absorbing mutations in the genome. Of the
mutations that occur in the genome, only inherited mutations play a vital
role in evolution or polymorphism. Thus repetitive DNA and mutational
forces functional in nature together form the basis of a number of marker
systems that are useful for various applications in plant genome analysis.
The markers belonging to this class are both hybridization-based and PCR-

Microsatellites and minisatellites

The term microsatellites was coined by Litt and Lutty, while the
term minisatellites was introduced by Jeffrey. Both are multilocus probes
creating complex banding patterns and are usually non-species specific
occurring ubiquitously. They essentially belong to the repetitive DNA
family. Fingerprints generated by these probes are also known as
oligonucleotide fingerprints. The methodology has been derived from RFLP
and specific fragments are visualized by hybridization with a labelled
micro- or minisatellite probe. Minisatellites are tandem repeats with a
monomer repeat length of about 11–60 bp, while microsatellites or short
tandem repeats/simple sequence repeats (STRs/ SSRs) consist of 1 to 6 bp
long monomer sequence that is repeated several times. These loci contain
tandem repeats that vary in the number of repeat units between
genotypes and are referred to as variable number of tandem repeats
(VNTRs) (i.e. a single locus that contains variable number of tandem
repeats between individuals) or hypervariable regions (HVRs) (i.e.
numerous loci containing tandem repeats within a genome generating
high levels of polymorphism between individuals).

Microsatellites and minisatellites thus form an ideal marker system

creating complex banding patterns by simultaneously detecting multiple
DNA loci. Some of the prominent features of these markers are that they
are dominant fingerprinting markers and codominant STMS (sequence
tagged microsatellites) markers. Many alleles exist in a population, the
level of heterozygosity is high and they follow Mendelian inheritance.
Minisatellite and microsatellite sequences converted into
PCR-based markers

Sequence-tagged microsatellite site markers (STMS)

This method includes DNA polymorphism using specific primers
designed from the sequence data of a specific locus. Primers
complementary to the flanking regions of the simple sequence repeat loci
yield highly polymorphic amplification products. Polymorphisms appear
because of variation in the number of tandem repeats (VNTR loci) in a
given repeat motif. Tri- and tetranucleotide microsatellites are more
popular for STMS analysis because they present a clear banding pattern
after PCR and gel electrophoresis. However, dinucleotides are generally
abundant in genomes and have been used as markers e.g. (CA)n(AG)n
and (AT)n (ref. 30). The di- and tetranucleotide repeats are present mostly
in the non-coding regions of the genome, while 57% of trinucleotide
repeats are shown to reside in or around the genes. A very good
relationship between the number of alleles detected and the total number
of simple repeats within the targeted microsatellite DNA has been
observed. Thus larger the repeat number in the microsatellite DNA,
greater is the number of alleles detected in a large population.

Direct amplification of minisatellite DNA markers (DAMD-PCR)

This technique, introduced by Heath et al., has been explored as a
means of generating DNA probes useful for detecting polymorphism.
DAMD-PCR clones can yield individualspecific DNA fingerprinting pattern
and thus have the potential as markers for
species differentiation and cultivar identification.

Inter simple sequence repeat markers (ISSR)

In this technique, reported by Zietkiewicz et al., primers based on
microsatellites are utilized to amplify inter-SSR DNA sequences. Here,
various microsatellites anchored at the 3¢ end are used foramplifying
genomic DNA which increases their specificity. These are mostly dominant
markers, though occasionally a few of them exhibit codominance. An
unlimited number of primers can be synthesized for various combinations
of di-, tri-, tetra- and pentanucleotides [(4)3 = 64, (4)4 = 256] etc. with an
anchor made up of a few bases and can be exploited for a broad range of
applications in plant species.

Other repetitive DNA-type markers

Transposable elements
A large number of transposable repeat elements have been studied
in plants; however, only a few have been exploited as molecular markers.
In evolutionary terms, they have contributed to genetic differences
between species and individuals by playing a role in retrotransposition
events promoting unequal crossing over. Retrotransposon-mediated
fingerprinting has been shown to be an efficient fingerprinting method for
detection of genetic differences between different species.
This strategy was developed to fingerprint genotypes using semi-
specific primers, complementary to repetitive DNA elements called ‘Alu-
repeats’, in human genome analysis. Alu repeats are a class of randomly
repeated interspersed DNA, preferentially used for Alu PCR as they reveal
considerable levels of polymorphism35. These representatives of short
and long interspersed nuclear elements are known as SINES. Alu elements
are approximately 300 bp in size and have been suggested to be
originated from special RNA species that have been reintegrated at a rate
of approximately one integration event per 10000 years. These repeats
have been studied largely in humans, while their function in plants
remains largely unexplored.

Repeat complementary primers

As an alternative to the interspersed repeats, primers
complementary to other repetitive sequence elements were also
successfully used for generation of polymorphisms, e.g. introns/exons
splice junctions, tRNA genes, 5sRNA genes and Zn-finger protein genes.
Primers complementary to specific exons, resulting in the amplification of
the intervening
introns have been studied by Lessa et al. One of the strengths of these
new strategies is that they are more amenable to automation than the
conventional hybridization-based techniques.
Arbitrary sequence markers
Randomly-amplified polymorphic DNA markers (RAPD)
In 1991 Welsh and McClelland developed a new PCR-based genetic
assay namely randomly amplified polymorphic DNA (RAPD). This
procedure detects nucleotide sequence polymorphisms in DNA by using a
single primer of arbitrary nucleotide sequence. In this reaction, a single
species of primer anneals to the genomic DNA at two different sites on
complementary strands of DNA template. If these priming sites are within
an amplifiable range of each other, a discrete DNA product is formed
through thermocyclic amplification. On an average, each primer directs
amplification of several discrete loci in the genome, making the assay
useful for efficient screening of nucleotide sequence polymorphism
between individuals42. However, due to the stoichastic nature of DNA
amplification with random sequence primers, it is important to optimize
and maintain consistent reaction conditions for reproducible DNA
amplification. They are dominant markers and hence have limitations in
their use as markers for mapping, which can be overcome to some extent
by selecting those markers that are linked in coupling. RAPD assay has
been used by several groups as efficient tools for identification of markers
linked to agronomically important traits, which are introgressed during the
development of near isogenic lines. The application of RAPDs and their
related modified markers in variability analysis and individual-specific
genotyping has largely been carried out, but is less popular due to
problems such as poor reproducibility faint or fuzzy products, and
difficulty in scoring bands, which lead to inappropriate inferences.

Some variations in the RAPD technique include DNA amplification

fingerprinting (DAF). Caetano-Anolles et al.44 employed single arbitrary
primers as short as 5 bases to amplify DNA using polymerase chain
reaction. In a spectrum of products obtained, simple patterns are useful as
genetic markers for mapping, while more complex patterns are useful for
DNA fingerprinting. Band patterns are reproducible and can be analysed
using polyacrylamide gel
electrophoresis and silver staining. DAF requires careful optimization of
parameters; however, it is extremely amenable to automation and
fluorescent tagging of primers for early and easy determination of
amplified products. DAF profiles can be tailored by employing various
modifications such as predigesting of template. This technique has been
useful in genetic typing and mapping.

Arbitrary primed polymerase chain reaction (AP-PCR)

This is a special case of RAPD, wherein discrete amplification
patterns are generated by employing single primers of 10–50 bases in
length in PCR of genomic DNA45. In the first two cycles, annealing is
under non-stringent conditions. The final products are structurally similar
to RAPD products. Compared to DAF, this variant of RAPD is not very
popular as it involves autoradiography. Recently, however, it has been
simplified by separating the fragments on agarose gels and using
ethidium bromide staining for visualization.
Sequence characterized amplified regions for amplification of
specific band (SCAR)
Michelmore et al. and Martin et al. introduced this technique
wherein the RAPD marker termini are sequenced and longer primers are
designed (22–24 nucleotide bases long) for specific amplification of a
particular locus. These are similar to STS markers48 in construction and
application. The presence or absence of the band indicates variation in
sequence. These are better reproducible than RAPDs. SCARs are usually
dominant markers, however, some of them can be converted into
codominant markers by digesting them with tetra cutting restriction
enzymes and polymorphism can be deduced by either denaturing gel
electrophoresis or SSCP. Compared to arbitrary primers, SCARs exhibit
several advantages in mapping studies (codominant SCARs are
informative for genetic mapping than dominant RAPDs), map-based
cloning as they can be used to screen pooled genomic libraries by PCR,
physical mapping, locus specificity, etc. SCARs also allow comparative
mapping or homology studies among related species, thus making it an
extremely adaptable concept in the near future.

Cleaved amplified polymorphic sequences (CAPs)

These polymorphic patterns are generated by restriction enzyme
digestion of PCR products. Such digests are compared for their differential
migration during electrophoresis49,50. PCR primer for this process can be
synthesized based on the sequence information available in databank of
genomic or cDNA sequences or cloned RAPD bands. These markers are
codominant in nature.

Randomly amplified microsatellite polymorphisms (RAMPO)

In this PCR-based strategy, genomic DNA is first amplified using
arbitrary (RAPD) primers. The amplified products are then
electrophoretically separated and the dried gel is hybridized with
microsatellite oligonucleotide probes. Several advantages of
oligonucleotide fingerprinting51, RAPD52 and microsatellite-primed PCR
are thus combined, these being the speed of the assay, the high
sensitivity, the high level of variability detected and the non-requirement
of prior DNA sequence information. This technique has been successfully
employed in the genetic fingerprinting of tomato, kiwi fruit and closely-
related genotypes of D. Bulbifera.

Amplified fragment length polymorphism (AFLP)

A recent approach by Zabeau et al.55, known as AFLP, is a
technique based on the detection of genomic restriction fragments by PCR
amplification and can be used for DNAs of any origin or complexity. The
fingerprints are produced, without any prior knowledge of sequence, using
a limited set of generic primers. The number of fragments detected in a
single reaction can be ‘tuned’ by selection of specific primer sets. AFLP
technique is reliable since stringent reaction conditions are used for
primer annealing. This technique thus shows an ingenious combination of
RFLP and PCR techniques and is extremely useful in detection of
polymorphism between closely related genotypes.

AFLP procedure mainly involves 3 steps

(a) Restriction of DNA using a rare cutting and a commonly cutting
restriction enzyme simultaneously (such as MseI and EcoRI) followed by
ligation of oligonucleotide adapters, of defined sequences including the
respective restriction enzyme sites.
(b) Selective amplifications of sets of restriction fragments, using
specifically designed primers. To achieve this, the 5' region of the primer
is made such that it would contain both the restriction enzyme sites on
either sides of the fragment complementary to the respective adapters,
while the 3' ends extend for a few arbitrarily chosen nucleotides into the
restriction fragments.
(c) Gel analysis of the amplified fragments.
AFLP analysis depicts unique fingerprints regardless of the origin and
complexity of the genome. Most AFLP fragments correspond to unique
positions on the genome and hence can be exploited as landmarks in
genetic and physical mapping. AFLPs are extremely useful as tools for
DNA fingerprinting58 and also for cloning and mapping of variety-specific
genomic DNA sequences. Similar to RAPDs, the bands of interest obtained
by AFLP can be converted into SCARs. Thus AFLP provides a newly
developed, important tool for a variety of applications.

Applications of molecular markers in plant genome

analysis and breeding

Molecular markers have been looked upon as tools for a large

number of applications ranging from localization of a gene to
improvement of plant varieties by marker-assisted selection. They have
also become extremely popular markers for phylogenetic analysis adding
new dimensions to the evolutionary theories. If we look at the history of
the development of these markers, it is evident that they have been
improved over the last two decades to provide easy, fast and automated
assistance to scientists and breeders. Genome analysis based on
molecular markers has generated a vast amount of information and a
number of databases are being generated to preserve and popularize it.

Mapping and tagging of genes: Generating tools for marker-

assisted selection in plant breeding
Plant improvement, either by natural selection or through the efforts
of breeders, has always relied upon creating, evaluating and selecting the
right combination of alleles. The manipulation of a large number of genes
is often required for improvement of even the simplest of characteristics.
With the use of molecular markers it is now a routine to trace valuable
alleles in a segregating population and mapping them. These markers
once mapped enable dissection of the complex traits into component
genetic units more precisely, thus providing breeders with new tools to
manage these complex units more efficiently in a breeding programme.
The very first genome map in plants was reported in maize, followed by
rice, Arabidopsis etc. using RFLP markers. Maps have since then been
constructed for several other crops like potato, barley, banana, members
of Brassicaceae, etc.

Once the framework maps are generated, a large number of

markers derived from
various techniques are used to saturate the maps as much as is possible.
Microsatellite markers, especially STMS markers, have been found to be
extremely useful in this regard. Owing to their quality of following clear
Mendelian inheritance, they can be easily used in the construction of
index maps, which can provide an anchor or reference point for specific
regions of the genome. About 30 microsatellites have already been
assigned to five linkage groups in Arabidopsis, while their integration into
the genetic linkage maps is still in progress in rice, soybean, maize, etc.
The very first attempt to map microsatellites in plants was made by Zhao
and Kochert68 in rice using (GGC)n, followed by mapping of (GA)n and
(GT)n by Tanksley et al.69 and (GA/AG)n, (ATC) 10 and (ATT) 14, by
Panaud et al in rice. The most recent microsatellite map has been
generated by Milbourne et al. for potato. Similar to microsatellites, looking
at the pattern of variation, generated by retrotransposons, it is now
proposed that apart from genetic variability, these markers are ideal for
integrating genetic maps.

Once mapped, these markers are efficiently employed in tagging

several individual traits that are extremely important for a breeding
programme like yield, disease resistance, stress tolerance, seed quality,
etc. A large number of monogenic and polygenic loci for various traits
have been identified in a number of plants, which are currently being
exploited by breeders and molecular biologists together, so as to make
the dream of marker-assisted selection come true. Tagging of useful
genes like the ones responsible for conferring resistance to plant
pathogen, synthesis of plant hormones, drought tolerance and a variety of
other important developmental pathway genes, is a major target. Such
tagged genes can also be used for detecting the presence of useful genes
in the new genotypes generated in a hybrid programme or by other
methods like transformation, etc. RFLP markers have proved their
importance as markers for gene tagging and are very useful in locating
and manipulating quantitative trait loci (QTL) in a number of crops. The
very first reports on gene tagging were from tomato, availing the means
for identification of markers linked to genes involved in several traits like
water use efficiency, resistance to Fusarium oxysporum (the 12 gene),
leaf rust resistance genes LR 9 and 24, and root knot nematodes
(Meliodogyne sp.) (the mi gene).